Lincomycin 3-nucleotides and the salts thereof

ABSTRACT

This invention relates to novel antibacterial compounds and to processes for preparing them. It is particularly directed to novel 3-nucleotides of lincomycin, and of analogs thereof, and of celesticetins. The compounds of the invention can be shown by the following formula:   WHEREIN Y is -SR wherein R is alkyl of one to six carbon atoms, inclusive, , OR -S-CH2-CH2-OH, R1 is H, or cis or trans lower-alkyl of from one to eight carbon atoms, inclusive; R2 is H, CH3, or C2H5; X is OH, chlorine, bromine, iodine or -OR3 wherein R3 is alkyl of one to six carbon atoms, inclusive, each in the (R) or (S) configuration; and Z is a nucleoside -5&#39;&#39;- phosphate group wherein said nucleoside can be adenosine, guanosine, cytidine or uridine; and salts thereof. Examples of alkyl of from one to eight carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl and isomers thereof.

United States Patent Argoudelis et al.

[54] LINCOMYCIN 3-NUCLEOTIDES AND THE SALTS THEREOF [72] Inventors: Alexander D. Argoudelis; John H. Coats,

both of Kalamazoo, Mich.

[73] Assign ee: The Upjohn Company, Kalamazoo, Mich.

[22] Filed: March 24, 1971 [2 l] Appl. No.: 127,852

Related U.S. Application Data [63] Continuation-in-part of Ser. No. 30,254, April 20,

l970, abandoned.

[52] U.S. Cl. ....260/2l0 R, 195/80, 424/180 Primary E.\'aminerLewis Gotts Assistant Examiner-Johnnie R. Brown Attorney-Roman Saliwanchik and John Kekich 5 7] ABSTRACT This invention relates to novel antibacterial compounds and to processes for preparing them. It is particularly directed to novel 3-nucleotides of lincomycin, and of analogs thereof, and

[ 1 June 20, 1972 of celesticetins. The compounds of the invention can be shown by the following formula:

O R HO /5 OZ A :3 2 Y wherein Y is SR wherein R is alkyl of one to six carbon atoms, inclusive,

, or S-CH -CH OH, R is H, or cis or trans lower-alkyl of from one to eight carbon atoms, inclusive; R is H, CH or C- H X is OH, chlorine, bromine, iodine or OR wherein R is alkyl of one to six carbon atoms, inclusive, each in the (R) or (S) configuration; and Z is a nucleoside 5' phosphate group wherein said nucleoside can be adenosine, guanosine, cytidine or uridine; and salts thereof.

Examples of alkyl of from one to eight carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl and isomers thereof.

19 Claims, No Drawings LINCOMYCIN S-NUCLEOTIDES ANDTHE SALTS THEREOF CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of co-pending application Ser. No. 30,254, filed Apr. 20, 1970 now. abandoned.

BRIEF SUMMARY OF THE INVENTION The novel compounds of the inventionare prepared by incorporating a compound, as defined in Formula I, wherein 2 at the 3-position of the molecule is hydrogen (hereinafter referred to as the parent" compound), in a Streptomyces fermentation, and transforming the compoundinto a novel 3- nucleotide, as described above. Also produced in varying amounts in the process of the subject invention, are 3- phosphate esters of the parent compounds. These 3-phosphate esters are readily distinguished from the 3-nucleotides of the subject invention since the 3-phosphates do not have. an ultraviolet absorption maximum and they are not hydrolyzed to the parent compound by snake venom diesterase (venom phosphodiesterase) or spleen diesterase. Thus, as will be described in detail hereinafter, the 3-nucleotides are detected in the recovery procedures of the subject invention by ultraviolet analysis and the snake venom diesterase test. Venom diesterase, cleaves, for example, clindamycin-adenylate, to clindamycin and adenosine-S '-phosphate.

Initial presence of S-nucleotides in fermentation beers is detected by the use of an alkaline phosphatase test, as hereinafter described. However, this test does not differentiate between 3-phosphates and 3-nucleotides and the 3- nucleotides would remain unrecognized but forthe use of other tests, as described above.

The compounds of the invention, though antibacterially inactive in vitro against S. aureus and Sarcina lutea,are activated when used in vivo, for example against S. aureus. Presumably, this activation in vivo, is comparable to the generation of the parent lincomycin compound when contacting the 3-nucleotide-lincomycin compound with alkaline phosphatase in vitro.

DETAILED DESCRIPTION Lincom'ycin With reference to Formula I,

wherein Y SCH, to -SC,H,, R cis or trans alkyl to 8 carbon atoms R, Hydrogen or alkyl to 8 carbon atoms X (S)OH or OR,

U.S. Pat. No. 3,086,9l2

U.S. Pat. No. 3,380,992 U.S. Pat. No. 3,380,992

U.S. Pat. No. 3,380,992

U.S. Pat; No. 3,380,992

X (R) or Belgium Pat. No. 676,202 (S) Cl or Br U.S. Pat. No. 3,496,163 X (R) or (S)| U.S. Pat. No. 3,496,163 Celesticetin U.S. Pat. No. 2,928,844 Desalicetin U.S. Pat. No. 2,85l,463

be prepared by the procedure disclosed in Examples 1 and 2 of U.S. Pat. No. 3,359,163 wherein said compound is named lincomycin C.

l'-Demethyl lincomycin, wherein Y is -SCH R is trans n-propyl, R, is H and X is (R)0H in Formula 1 can be prepared by the procedure disclosed in Example I of U.S. Pat. No. 3,329,568 wherein said compound is named lincomycin D.

Of the above compounds, the compound 7(S)-chloro 7- deoxylincomycin is also presently known by the generic name clindamycin.

The parent lincomycin compounds or analogs thereof, and celesticetin, as described above, can be converted to 3 nucleotides, as shown in Formula I, by incorporating the parent compound in a Streptomyces fermentation. For example, upon adding clindamycin hydrochloride to a Streptomyces coelicolor Miiller, NRRL 3532, fermentation there are produced clindamycin nucleotides.

The fermentation to make the novel compounds of the invention can be conducted in an aqueousnutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts of 3-nucleotides, surface cultures and bottles can be employed. The organism used in the fermentation is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include corn steep liquor, yeast, autolyzed brewers yeast with mil solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers, solubles, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbons and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron and the like need not be added to the fermentation media since tap water and unpurified ingredients are used as media components.

Production of the novel compounds of the invention can be effected at any temperature conducive to satisfactory growth of the Streptomyces culture, for example, between about 18 and 40 C., and preferably between about 20and 37C.

When a Streptomyces fermentation, as described above, is used to prepare nucleotides of lincomycin or an analog thereof, as herein defined, or of celesticetin, the lincomycin or celesticetin parent compound (non-nucleotide) can be added prior to inoculation of the fermentation medium. Alternatively, the parent compound can be added in small increments during the fermentation cycle so long as the addition is not too late in the fermentation cycle to accomplish the desired transformation of all the parent compound added. The time and amounts of addition of the parent compound can easily be determined for each fermentation by adding the parent compound until some toxicity to the-fermentation is observed, such as inhibition of the formation of 3-nucleotides. Also, if at the end of the fermentation cycle there remains parent compound, then in subsequent fermentations smaller levels of parent compound should be used and/or the time of addition should be altered.

Since the in vitro antibacterial activity against S. lutea of the parent compound is lost upon transformation to a 3-nucleotide, the presence of residual in vitro antibacterial activity in a culture or culture extract at 24 hours after addition of the parent compound is evidence that the capacity of the culture or culture extract to transform the parent compound has been exceeded or the level or added compound was too high and inhibited the microorganism in the transformation process. The in vitro antibacterial activity, mentioned above, can be ascertained on a standard microbiological plate assay against the microorganism Sarcina Iutea.

A variety of procedures can be employed in the isolation and purification of the novel compounds in the subject invention, for example, solvent extraction, liquid-liquid distribution in a Craig apparatus, liquid ion exchange extraction or adsorption on a suitable adsorbent, for example, carbon, and column chromatography. In a preferred recovery process, the novel 3- nucleotide compounds are isolated from a fermentation beer, as herein described, by filtration. The filtrate is then passed over a suitable absorbent, for example, activated carbon or Amberlite XAD-Z (a non-ionic, macro-porous copolymer of styrene cross-linked with divinylbenzene resin sold by Rohm and Haas Company). This resin is prepared by suspension polymerization of styrene divinylbenzene copolymers in the presence of a substance which is a good solvent for the copolymer (see J.A.C.S. 84, 306,1962) to remove water-soluble impurities which may interfere with the subsequent chromatography step. The resin is eluted with a mixture of water and water-miscible organic solvents, for example, water-lower alcohols, water-lower ketones, and the like. The eluate from the carbon or Amberlite XAD-2 resin is then passed through a chromatography column containing an anion exchange resin, for example, Dowex-l (X- 4) in the acetate form (sold by Dow Chemical Company, Midland, Michigan). Fractions are collected from the chromatography column and assayed for activity against the microorganism S.lutea before and after treatment of the fractions with alkaline phosphatase as hereinafter described. Fractions having the highest activity against S. lutea upon test with alkaline phosphatase are pooled, concentrated, then subjected too countercurrent distribution in a Craig apparatus using a solvent system consisting of n-butanol-water (1:1 v/v). Fractions showing maximum ultraviolet absorption, and which are hydrolyzed by snake venom phosphodiesterase, are collected to give a preparation containing a mixture of 3-nucleotides. This mixture can be subjected to separation procedures to recover the individual S-nucIeotides.

A preferred separation procedure to recover the individual 3-nucleotidesfrom a mixture thereof utilizes DEAE-Sephadex (Pharmacia Fine Chemicals, Inc., Piscataway, N.J., U.S.A. or Pharmacia, Uppsalu, Sweden) column chromatography. The column is eluted with tris-(hydroxymethyl) amino-methane (THAM) acetate. Fractions are analyzed by testing for activity against S lutea before and after alkaline phosphatase treatment and by the ultraviolet spectrum analysis at the original pH of the fraction, and at an acid pH (ca. 2.0). Pools of fractions having biological activity against S Iutea after alkaline phosphatase treatment, and showing ultraviolet spectrum absorption, are made. Each pool contains a single 3-nucleotide, along with other undesired materials. The THAM-acetate buffer is removed'from these pools by passing them over a column containing Amberlite XAD-2 packed in water. After washing the column with water, it is eluted with aqueous methanol (ca. 80 percent aqueous methanol). Fractions, about 20 ml. each, are collected and analyzed by U.V. Fractions showing U.V. absorption are combined and concentrated to dryness to a residue. The residue is dissolved in a lower alcohol, for example methanol, and the solution mixed with ether to yield a precipitate of a 3-nucleotide compound, as defined in Formula I. As shown in Formula I, the 3-nucleotide moiety of the novel compounds of the invention are the 3- (5 '-cytidylate), 3-(5'- adenylate), 3-(5'-uridylate), and 3-(5'- guanylate).

The nucleotides can also be separated by partition chromatography over Dicalite (diatomaceous earth) using solvent systems consisting of water and a water-immiscible solvent.

Lincomycin 3-nucleotides and the 3-nucleotides of lincomycin analogues are essentially inactive against bacteria in vitro. Thus, these novel 3-nucleotide compounds are detected the novel compounds of the invention are S. coelicolor 1945,

NRRL 3531; S. caelicolor Miiller, NRRL 3532; and S. venezuelae, NRRL 3527. These cultures are avaiiasrawimom restriction, from the Northern Utilization and Research Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, U.S.A.

The novel compounds of the invention are amphoteric compounds and can exist in different ionic forms according to the pH of the environment. At low pH the compounds exist in the acid-addition salt form, at a higher pH in a zwitterion form, and at a still higher pH in a metal salt form. The acid-addition salts include those of strong organic or inorganic acids having a pK equal to or less than that of phosphate, for example, hydrochloric, sulfuric, phosphoric, and like acids.

Acid and metal salts include the alkaline metal (Na and K as well as including ammonia) and alkaline earth metal (including calcium, magnesium, zinc and aluminum) salts obtained by neutralizing an acid form with the appropriate base, for example, ammonium hydroxide, sodium and potassium hydroxides, or alkoxides, calcium, or magnesium hydroxides, and the like. The acid and neutral salts also include amine salts obtained by neutralizing an acid form with a basic amine, for example, mono-, di-, and trimethylamines, mono-, di-, and triethylamines, mono-, di-, and tripropylamines (isoand normal), ethyldimethylamine, benzyldiethylamine, cyclohexylamine, benzylamine, dibenzylamine, N ,N dibenzylethylenediamine, bis-( ortho-methoxyphenylisopropyl)amine, and like lower-aliphatic, lowercycloaliphatic, and lower-araliphatic amines, the loweraliphatic and lower-cycloaliphatic radicals containing up to and including eight carbon atoms; heterocyclic amines such as piperidine, morpholine, pyrrolidine, piperazine, and the lower-alkyl derivatives wherein lower alkyl contains 1 and 8 carbon atoms, inclusive thereof such as l-methylpiperidine, 4- ethylm orpholine, l-isopropylpyrrolidine, l ,4-dimethylpiperazine, l-n-butylpiperidine, 2-methylpiperidine and lethyl-2-methylpiperidine; amines containing water solubilizing or hydrophilic groups such as mono di-, and triethanolamines, ethyldiethanolamine, n-butyl monoethanolamine, 2-amino-lbutanol, 2-amino-2-ethyl- 1 ,3- propanediol, 2-amino-2-methyl-l-propanol, tn's(hydroxymethyl)-aminomethane, phenylmonethanolamine, p-tertiaryamylphenyldiethanolamine, and galactamine, N -methylglucamine, N-methylglucosamine, ephedrine, phenylephrine, epinephrine, and procaine; tetraethylammonium hydroxide; and guanidine. The various forms can be used interchangeably but for most purposes the zwitterion form Re Ha l 029 l if wherein R R,, X, Y and Z are as defined previously, and the ammonium salt form are preferred.

Further, the invention relates to a process for the therapeutic treatment of humans and animals hosting susceptible microbial disease-producing organisms (bacterial and other microparasites) and the prophylactic treatment of a diseasesusceptible host comprising the administration of the 3- nucleo-tide esters or a pharrnacologically acceptable salt to the host. 1

The compositions of the present invention are useful in the same manner as lincomycin and celesticetin in the treatment of humans, birds, and animals for various pathological conditions. The compositions provide a means for administering the therapeutic ingredient by the oral and parenteral routes for systemic treatment. The compositions provide a method of therapy for tonsillitis, pneumonia, otitis, conjunctivitis, boils, carbuncles and other infectious conditions of humans due to the presence of bacteria. in animals, the compositions can be used prophylactically. For example, rats can be protected from Streptococcus viridans during shipment. Animals raised for meat can be given prophylactic treatment for increased weight gain.

Mammals hosting a parasitic protozoan of the class Sporazoa, order Coccidia a microparasite producing the disease coccidiousis) can be treated by administration of the compositions of the present invention. For example cattle infected with E. zurnii, E. bovis, E. illiprordalis; sheep and goats with E. parva, E. fourei; swine with E. debliecki, E. scabra, and Isospora suic; dogs and cats with Isospora biqemina, Isospora felis, E. canis, E. felini; poultry with E. tenella; rabbits with E, steedae, E, perforans; and mink with E. mustelae can be treated.

The compositions are also useful in the treatment of diseases caused by members of the genus Mycoplasma, the most commonly known forms are PPLO (pleuropneumonia-like organisms) such as M. hominis, M. salivarium, M. mycoides, M. hyopneumonia, M. hyorhinis, M. gallisepticum, M. arthriditis and other species in man and animals, including domestic animals such as sheep, dogs, cattle, swine, and poultry (e.g., chickens, turkeys, ducks, and geese) and laboratory animals (e.g., rats and mice).

The compositions find application in the treatment of kidney and other infections when L forms of gram-negative and gram-positive bacteria are present, for example, L forms of P. mirabilis.

The 3-nucleotides and salts disclosed herein are presented for oral and parenteral administration in solid and liquid unit dosage forms, such as tablets, capsules, powders, granules, pills, sterile parenteral solutions and suspensions, and oral solutions and suspensions, and oil-water emulsions.

Powders are prepared by comminuting the 3-nucleotides to a suitably fine size and mixing with a similarly comminuted diluent. The diluent can be edible carbohydrate material such as starch or lactose. Advantageously, a sweetening agent or sugar is present as well as a flavoring material. Dry granulations for reconstitution with water are prepared utilizing water-soluble diluents. A powder mixture of a finely divided 3- nucleotide and a water-soluble diluent such as sucrose, glucose, and the like, is wetted with a binder such as acacia mucilage or gelatin solution and forced through a screen to form granules which are allowed to dry. Advantageously, a thickening or suspending agent such as methylcellulose is present as well as a wetting agent and flavoring oil.

Capsules are produced by preparing a powder mixture as hereinbefore described and filling into formed gelatin sheaths. Advantageously, as an adjuvant to the filling operation, a lubricant such as talc, magnesium stearate and calcium stearate is added to the powder mixture before the filling operation.

Tablets are made by preparing a powder mixture, wet granulating or dry granulating or slugging, adding a lubricant, and pressing into tablets. The powder mixture is prepared by mixing the 3-nucleotide suitably comminuted, with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The powder mixture can be granulated by wetting with a binder such as corn syrup, gelatin solution, methylcellulose solution or acacia mucilage and forcing through a screen. As an alternative granulating procedure, the powder mixture can be slugged, i.e., run through the tablet machine and the resulting large tablets (slugs) broken into granules. The granules can be lubricated to prevent sticking to the tablet-forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. Advantageously, the tablet can be provided with a protective coating consisting of a sealing coat of shellac, a coating of sugar and methylcellulose, and a polish coating of carnauba wax.

Oral fluids are prepared in unit dosage forms such as syrups and elixirs wherein each teaspoonful of composition contains a predetermined amount of the 3-nucleotide for administration.

A syrup is prepared by dispersing the 3-nucleotide in a suitable flavored aqueous sucrose solution. Similarly, an elixir is prepared utilizing a hydro-alcoholic vehicle. Elixirs are advantageous vehicles for use when a solution is desired of a compound showing low solubility in water and good solubility in an aqueous-alcoholic medium.

For parenteral administration, sterile fluid unit dosage forms can be prepared. In preparing the parenteral form, a measured amount of the S-nucleotide is placed in a vial; the vial and its contents sterilized and sealed. An accompanying vial of sterile water can be conveniently provided as a vehicle to form a suspension or solution (depending on water-solubility of compound) prior to administration. Advantageously, the sterile water can have dissolved therein a suspending agent, local anesthetic, and buffering agents.

Alternatively, a parenteral suspensionhaving prolonged action can be prepared by suspending the 3-nucleotide in a parenterally acceptable vegetable oil with or without additional adjuvants.

The term unit dosage form as used in the specification and claims refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to the desired dosage in association with the required pharmaceutical diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for therapeutic use as disclosed in detail in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, powder packets, granules, wafers, teaspoonfuls, tablespoonfuls, dropperfuls, ampuls, vials, segregated multiples of any of the foregoing, and other forms as herein described. The unit dosage forms compounded with a suitable phannaceutical carrier contain, in the preferred embodiment, from 25 mg. to 500 mg. of 3-nucleotide or its pharmacologically acceptable salts per dosage unit and 5 to 65 percent w/v for parenteral preparations.

The amount of 3-nucleotide or salts thereof that is to be administered depends on the age and weight of the patient, the particular condition to be treated, and the route of administration. A dose of from about I mg.lkg./day to about 50 mg./kg./day is preferred for systemic treatment.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 CLlNDAMYClN-3-NUCLEOTIDES A. Fermentation A soil stock of Streptomyces coelicolor Mllller, NRRL 3532, is used to inoculate a series of 500-ml. Erlenmeyer flasks, each containing ml. of sterile seed medium consisting of the following ingredients:

Glucose monohydrate 25 g./liter Pharmamedia" 25 gJliter Tap water q.s. Balance 'Pharmamedia is an industrial grade of cotton-seed flour produced by Trader's Oil Mill Company, Fort Worth, Texas.

2.5 g./liter 5.0 gJliter Yeast Extract NZ amine B Glucose monohydrate 20.0 g.lliter Sodium nitrate 3.0 g./liter Dipotassium phosphate 1.0 g.lliter Magnesium sulphate 0.5 g.lliter Potassium chloride 0.5 g./liter Ferrous sulphate 0.01 g.lliter Tap water Balance Sold by Difco Laboratories, Detroit, Michigan. It is a bulk peptone in powder form obtained by the pancreatic digestion of casein.

100 mg/liter clindamycin hydrochloride is added to the fermentation flask broth 24 hours after inoculation.

The fermentation flasks are grown for 24 hours at 28 C. on a rotary shaker. The transformation reaction in the fermentation flask is followed by measuring the loss of clindamycin activity using an S. lutea standard curve assay. Approximately 100 percent of the added cylindamycin is transformed to an in vitro antibacterially inactive form in about 24 hours. The S. Iutea assay is conducted as follows: The assay is on agar buffered to pH 6-8 with pH 7.0 phosphate buffer (0.1 M). A unit volume (0.08 ml.) of solution containing the material to be assayed is placed on a 12.7 mm. assay disc which is then placed on an agar plate seeded with the assay microorganism. The tray is incubated at 37 C. for 18-24 hours. In vitro antibacterial activity is evidenced by a zone of grown inhibition surrounding the disc. Antibacterial activity can be expressed quantitatively as meg. parent compound (or as lincomycin or clindamycin) /ml. by the linear relation of log dose to zone diameter, referred to the standard, according to the art. Presence of clyindamycin-3-nucleotides is determined by first incubating the inactive beer with alkaline phosphatase at pH 8.0 in Tris buffer, and then assaying the reaction mixture against S. lutea, as described above. B. Recovery l. Filtration and Absorption on Non-Ionic Resin The above fermentation is scaled up into a fermentation tank to produce 490 liters of fermentation beer containing clindamycin-3-nucleotides. The clindamycin-3-nucleotides are recovered from the whole beer by first filtering the whole beer with the aid of 10 kg. diatomaceous filter aid. The filter cake is washed with water. The aqueous wash is combined with the clear beer and the combined clear beer-wash is treated with an adsorbent, for example carbon or Amberlite AXD-2 (sold by Rohm and Haas Company), in order to remove water-soluble impurities which tend to reduce the efficiency of subsequent chromatography. The absorption column is prepared by slurrying about 22 kg. of adsorbent (Amberlite XAD-2) in water, pouring the slurry into a glass column (2 inches inside diameter), allowing the slurry to settle under atmospheric pressure, and draining. The clear beerwash, described above, is passed through the column at a flow rate of about 1 liter per minute. The column is washed with water; 100 liters of the water wash is discarded. The column is then eluted with 120 liters of 60 percent aqueous methanol (Eluate l) and 100 liters of 95 percent aqueous methanol (Eluate ll). Eluate l is treated further to recover clyindamycin-3- nucleotides, whereas Eluate 11 is discarded.

2. Absorption on lon Exchange Resin Eluate I, described above, is chromatographed over an anion exchange chromatographic column. The column is filled with 22 Kg. Dowex-l (X-4) in the acetate form, supplied by Dow Chemical Company, Midland, Michigan. Eluate l is adjusted to a pH of 10.0 with concentrated ammonium hydroxide and the alkaline solution is passed through the chromatography column. The spent liquid from the column is concentrated to dryness; yield, 877 grams of material containing clindamycin-3-nucleotides. This material is labeled Material A. The column is then washed with 100 liters of water and eluted with 70 liters of 5 percent aqueous acetic acid. The acetic acid elutes are concentrated and the resulting concentrate is freeze-dried; yield 89.4 grams of material containing clindamycin-3-nucleotides. This material is labeled Material B."

C. Purification 1. Absorption on Non-Ionic Resin A major portion (776 g.) of Material A, obtained as described above, is dissolved in 1.5 liters of water. The pH of the solution is adjusted to 7.5 with concentrated ammonium hydroxide and this solution is passed over a column containing 2 liters of Amberlite XAD-Z resin. The column is washed with 6 liters of water. The aqueous wash is collected in three 2-liter fractions (W-l, W-2, W-3). The column is then eluted with percent aqueous methanol. Fractions of 20 ml. are collected and tested for activity against S. Iutea before and after treatment with alkaline phosphatase. Fractions numbered 61-250 are combined and concentrated to dryness; yield, 52 g. of material containing cylindamycin-3-nucleotides. This material is labeled ADA-10.1."

Fractions W-2 and W-3, described above, and fractions numbered 1-60 from the above Amberlite XAD-2 column, are combined and passed again over the same Amberlite XAD-2 column, which is first regenerated with 15 liters of water (W-l obtained as described above) and then eluted with 4 liters of absolute methanol. Three cuts are made, i.e. methanol fraction 1 1 liter; methanol fraction 2 1 liter; and, methanol fraction 3 3 liters. These fractions are tested for activity against S. [area before and after treatment with alkaline phosphatase. Methanol fraction 2 is concentrated to dryness; yield, 12.63 g. of material containing clindamycin-3- nucleotides. This preparation is labeled ADA-1 1.1."

Methanol fraction 3 is concentrated to dryness; yield, 0.7 g. of material containing clindamycin-3-nucleotides. This preparation is labeled ADA-1 1.2."

Preparations ADA-10.1 ADA-11.1, and ADA-11.2, all prepared as described above, are combined as preparation ADA-37.1 (64.7 g.). This preparation containing clindamycin-3-nucleotides is purified further by counter double current distribution as described below.

2. Counter Double Current Distribution A portion (21 g.) of preparation ADA-37.1, described above, is dissolved in ml. of upper and 100 ml. of the lower phase of a solvent system consisting of n-butanol-water (1:1 v/v). The solution is added in the center tubes of an allglass counter double current distribution apparatus (CDCD) (100 tubes). After 48 transfers, both the upper and lower phase are collected in 50 ml. fractions. A total of 100 transfers are run. The collected fractions and the material in the CDCD tubes are analyzed for S. lutea activity before and after treatment with alkaline phosphatase.

Using the same conditions as above, two additional CDCD distributions are run, each using 21 g. of preparation ADA-37.1.

in each of the above three distributions the following pools of fractions are made:

Pool l Lower-phase collec tor- Fractions number 20-50.

Pool 11 Lower and upper-ph ase remaining in the CDCD machine.

Pool lll Upper-phase collec tor- Fractions numbered 5-35.

Pool I from all three distributions are concentrated to dryness. The resulting residue is dissolved in absolute methanol and this solution is mixed with ether. The resulting precipitate is isolated by filtration and dried; yield, 7.12 g. This preparation is not pursued further.

Pools 11 and ill from all three distributions are treated as above for Pool 1; yield, 13.6 g. of material from Pool 11 labeled ADA-39.2," and 0.49 g. of material from Pool lll labeled ADA-39.3." Preparations ADA-39.2 and ADA-39.3 consist of essentially pure clindamycin-3-nucleotides as evidenced by inactivity against S. [area before treatment with alkaline phosphatase, and activity against S. lutea after treatment with alkaline phosphatase. The presence of clindamycin after phosphatase treatment is shown by TLC (Thin-layer chromatography).

D. Separation of Clindamycin-3-Nucleotides by Chromatography The clindamycin-3-nucleotides, obtained as described above, are separated into the individual clindamycin-3- nucleotides by use of DEAE Sephadex Chromatography. The resin is prepared by slurrying 500 g. of DEAE Sephadex (A-25) with water for about one hour. The resin is separated by filtration and stirred with 0.5 N aqueous sodium hydroxide solution for 2 hours. The resin is again isolated by filtration and washed with water until the pH of the wash is almost neutral. The washed resin is then stirred with 0.5 N aqueous acetic acid for 2 hours, and finally washed to a neutral pH.

The resin, prepared as described above, is added into a glass column and allowed to settle under atmospheric pressure. The column is washed with 4 l. of water and then with 4 1. of 0.1 percent aqueous solution of tris-(hydroxymethyl)- aminomethane (Tl-1AM).

The starting material (ADA-39.2, 13.0 g.) is dissolved in 100 ml. of water. The pH is adjusted to 9.0 with concentrated ammonium hydroxide. This solution is then added on the top of the column. The column is eluted sequentially as follows:

1. l. of 0.05 M THAM acetate (prepared by dissolving 6.05 g. of THAM in 800 ml. of water, adjusting the pH to 8.0 with glacial acetic acid and then adjusting the volume to 1 1.).

2. 40 l. of 0.1 M THAM acetate buffer, pH 8.0. 3. 1. of 0.2 M THAM acetate buffer, pH 8.0. I

4. 20 l. of0.3 M THAM acetate buffer, pH 8.0.

Fractions of 20 ml. are collected. The following fractions are obtained from each buffer:

From 0.05 M buffer, fractions 1-722 From 0.1 M buffer, fractions 723-2920 From 0.2 M buffer, fractions 2921-3985 From 0.3 M buffer, fractions 2985-5000 Selected fractions are analyzed by testing for activity against S. Iutea before and after alkaline phosphatase treatment and by U.V. spectra of the effluent of the column both as it is obtained and at acid pH (ca. 2.0).

The following pools are made:

Pool 1 Fractions: 850-965 Volume: ca. 2,300 ml.

A max. (a) U.V.: neutral, pH 7.0 270(3.72) acid, pH 2.0 279(5.40) base, pH 11.0 271(3.72)

Pool 11 Fractions: 1,2401,535 Volume: ca. 5,200 ml.

A max. (a) U.V.: neutral, pH 7.0 261(11.4) acid, pH 2.0 255(11.25) base, pH 11.0 258(1125) Pool 111 Fractions: 1,5501,680 Volume: 2,600 ml.

A max. (a) U.V.: neutral, pH 7.0 2620.60) acid, pH 2.0 2620.64) base, pH 11.0 261(2.82)

Pool 1V Fractions: 1,771-2,125 Volume: 7,000 ml.

A max. (a) U.V.: neutral, pH 7.0 2540.74): sh 278 acid, pH 2.0 254(3.66): sh 278 base, pH 11.0 264(3.20)

a. Isolation of C1indamycin-3-(5 '-Cytidylate Present 1n P0011 By Chromatography The column is preparedfrom 150 ml. of Amberlite XAD-2. Pool 1, prepared as described above, is passed over the column at a rate of 6 m1./min. The spent is collected in 116 20-m1. fractions. All fractions show no U.V. maximum and are discarded. The column is then washed with 900 ml. of water (fractions 117-161). The wash is also discarded. The column is then eluted with percent aqueous methanol. Fractions are analyzed by U.V. Results follow:

evaporated to an aqueous concentrate and freeze-dried; yield, 750 mg. of clindamycin-3-(5 cytidylate).

Five hundred mg. of this preparation is dissolved in 5 m1. of methanol and the solution is mixed with ether; yield, 400 mg. of c1indamycin-3-(5'-cytidylate), having the following structure:

I (it Analytical data Calcd. for: C,-,H,,,N,O,,PSC1: C, 44.48, H, 6.17; N, 9.60; O, 26.39; S, 4.39; CI, 4.87; P,

4.25. Found:

C, 45.62; 45.86; H, 6.99; 7.63; N, 9.80; S, 3.61; CI, 3.90;

1 Fraction No. A max. (a) Molecular weight Calcd. for: C -,H, N O,,PSCI: 729.5 s 260 NO maximum Found! 5 260 (0.18) 742 (vapor pressure osmometry, in methanol). 12 260 (0.46) 14 260 0.47 15 260 (230.0) Potentlometrlc titration I6 0 32 In water: pKa' 7.7 17 260 (628) eq. wt. 587 10 13 260 (540) 19 260 (405) Specific Rotation: y 22%(328) [a],, ,+61(c, l,water) 22 2602135; infrared Spectrum: 23 260 (30) The infrared spectra in both mineral oil mull and K131 pellet 24 260 (55) are as follows: 25 (3 I 5) 26 260 (26.4) 27 260 (12.8) IN MINERAL OIL MULL 2 260 (9.0) 29 260 (5.5)

1 i so 260 (3.65)

1'0 f0 H H r l iten- ((monug I ztcnuenc y) Ifiteng; I till l'tEllllillt'y (1111. my 0111. S1 111. S

y b y 33 260 (1.55) 11,340 8 1,400 8 092 3 3,210.. s 11,455 3 067 M 2,030 s 1 1,375 s 033 M 2,800 S 1 1,305 s 886 8 2,700 M 1,28). s 853 M 1:- 1' 121 1,1'.1 1, 05 s 1,010 a 1,070 8 720 s Fractions 15-21 are combined. The solution 1s mixed w1th v 9 f 1,500 ml. of acetone. The precipitated material is collected [11 Km P011012 and dried; yield, 2.1 g. of clindamycin-Z-(S'-adenylate) having :1,100 s 7 1,500 M "m 1,045 the followmg Structure: :1,210 s 1,520 s 000 M 11,100 0' 1,4110 s 005 M 2,055, s 1,455 M 030 M 2,025 8 1,305 M 88). M on 2,1170 M 1,3140 M 1150 M l 3 on 2,700 M 1,2011 M 2 1100 M N I 3 1,040. s 1,215 s 7115 M 1,1115, s 1,0101 s 700 M R] 1,575 M 1,005 s I NII( II II I (111. -0 2 S11. 0 "T I I oz Band 1ntens1t1es 1n the IR. spectra, disclosed herein, are m- 1 0H m dicated as S, M, and W, respectively, and are approximated in R1=CII2C 11 C11 terms of the backgrounds in the vicinity of the bands. An S IIIHI band is of the same order of intensity as the strongest in the spectrum; M bands are between one-third and two-thirds as N intense as the strongest band, and W bands are less than onethird as intense as the strongest band. These estimates are \N/\N made on the basis of a percent transmission scale. The Z=P0C H1 designation (sh)" refers to a shoulder.

so I O U.V.Spcctrum: in water at the follow ing pHs:

(LH H A max. a 1 pH 2.0 279 l3.l6 9,600 pH 7.0 269 9.37 6,835 pH ll.0 27l 9.10 6,638

Clindamycin-3-(5 '-adenylate) has the following chemical Reactions wlth Enzymes and physica properties: Crude Alkaline Phosphatase Analytical data Treatment with alkaline phosphatase yields clindamycin 6o Calm forr u a 1 u identified by thin-layer chromatography (silica gel, ethy 1 1 1 1397; K 1 acetate-acetone-water (825:1 v/v)). Found: venom Dieste ase C, 44.77; H: 6.66; N, 12.57; S, 4.65; C], 4.38; P, 3.52.

Treatment with venom diesterase yields clindamycin Molecular welght identified by thin-layer chromatography (as above). In addi- 65 Calci aa u -l n 753-5 tion to clindamycin, cytidine-5'-phosphate is produced. FOund! 726 (vapor pressure osmometry, in methanol) b. Isolation of Clindamycin-3-( 5 '-Adenylate) Pool [I by Chromatography Potentiometric titration in water: pKa' 7.6 The column is prepared from 400 m1. of Amberllte XAD-Z. Eq. wt. 620 Pool 11 is passed over the column at a flow rate of 15 ml./min. S ifi rotati The column is washed with 4 l. of water. Both spent and aque- [011 +62.9 (c, 1.04, water) ous wash do not show U.V. maxima and are discarded. The infrared spectrum: column is eluted with 80 percent aqueous methanol. Fractions The infrared spectra in both mineral oil mull and KBr pellet are analyzed by U.V. Results follow: are as follows:

In Mineral Oil Mull Band Band frcfre- Band frequency Intenquency Intenquency Inten- (umr sity (cmr sity (crnr slty 1,470 s 1,065 s 2 1,450 S 1,050 S 1 1,445 S 987 S 1 1,435 S 965 S 1,417 S .127 M 2 1,373 S 885 S 1 1,363 S 853 M 1,327 S 817 M 1,208 S 705 S 1 1,?A5- b a 717 S In K111 pullol.

1,037 S 1,313 3 1,503 B 1,085 S 1,570 M 1,065 s 1,510 M 1,045 s 1,470 M 986 M 1,453 M 027 M 1,415 M 885 M 1,375 M 852 M 1,325 M 815 M 1,295 M 705 M 1 1,245 M 717 M l Sh. 2 Oil. U.V. Spectrum: ln water at the follo wing pHs:

max. 5 pH 2.0 257 16.76 12,628 pH 7.0 261 16.67 12,560 pH 11.0 261 16.87 12,711

Reactions with Enzymes in Vivo Activity Clindamycin-3-(5'-adenylate) does not possess in vitro antibacterial activity against S. lutea. However, it is active in vivo (mice, S.Q., S. aureus) with a CD of 30 mg./kg.

c. Isolation of Clindamycin-3-(5'-Uridylate) Present in Pool ill by Chromatography The column is prepared from 150 ml. of Amberlite XAD-2. Pool lll is passed over the column at a rate of ml./min. The column is washed with l l. of water. Both the spent and the aqueous wash do not show U.V. maximum. The column is then eluted with 80 percent aqueous methanol. Fractions are analyzed by U.V. Results follow:

Fraction No. A max. (at) No maximum 261 (0.25) 261 (0.97) 261 (107 261 (248) 261 ('75) 261 (18) 261 (2.5) 261 (0.87)

oooqasv-e-w Fractions 6-9 are combined. The solution is concentrated to dryness. The residue is dissolved in methanol and this solution is mixed with ether to give a precipitate; yield, 740 mg.-. of clindamycin-3-(5'-uridylate) having the following structure:

| on: N I n- -01 R; OONlI- II T l)/s om Clindamycin-3-(5-uridylate) has the following chemical and physical properties: Analytical data Calcd. for: C H ND PSCI;

C, 44.27; H, 6.33; N, 7.68; O, 28.49; S, 4.39; CI, 4.86; P,

Found: C, 44.62; H, 6.19; N, 7.79; S, 4.04; CI, 4.32; P, 4.22. Molecular weight Calcd. for C -,H N O,=,PSCl: 732.5 Found: 764 (vapor pressure osmometry in methanol) Potentiometric titration In Water: pKa', 7.6

Eq. WL, 576

Specific Rotation [a],, +79.5 (c, 0.99, water) Infrared Spectrum The infrared spectra in both mineral oil mull and KBr pellet are as follows:

IN MINERAL OIL MULL Band Band frofre- Intcnqnenvy Intonqunncy Inton- Bantl frequency (0111:) sity (mnf sity (01117) sity l 1, 060 t 1, 000 S l, 515 S .190 S l, 515 S 885 h 2 1, 455 S 850 S 2 1.375 S 810 t 1, s 763 s l, 215 S 2 720 S In K131 Pellet 1,685 S .100 .\1 1,512 M .165 .\I 1,458 N 883 M 1,380 M 850 M 1, 2.00 S 808 .\l 1, 215 S 760 M 1,085 S 705 M -1, 060 S 1 Sh. (Oil. 7 H 7 V U.V. Spectrum: lnwater at the follo wing pH s:

A max. '0 6 pH 2.0 261 11.18 8,189 pH 7.0 262 l 1.44 8,379 pH ".0 262 8.90 6,519

Reaction With Enzymes Crude Alkaline Phosphatase Treatment with alkaline phosphatase yields clindamycin identified by thin-layer chromatography (silica gel, ethyl acetate-acetone-water(8:511 vlv)). Venom Diesterase Treatment with venom diesterase yields clindamycin and uridine-S '-phosphate.

In Vivo Activity Clindamycin-3(5'-uridylate) does not possess antibacterial activity against Sarcina lutea in vitro. However, his active in vivo (8.0., mice, S. aureus) with a CD of 37 mgJkg.

d. Isolation of Clindamycin-3-(5 '-Guanylate) Present in Pool IV by Chromatography The column is prepared from 200 ml. of Amberlite XAD-Z. Pool IV is passed over the column at a rate of ml./min. The column is washed with 3 l. of water. Both spent and aqueous wash show no U.V. maximum. The column is eluted with 80 percent aqueous methanol. Fractions are analyzed by U.V. Results follow:

Fraction No. A Max. (a)

2 No maximum 4 No maximum 6 254 (0.56); sh 278 7 254 (260); sh 278 8 254 (740); sh 278 9 254 (400); sh 278 I0 254(168);sh 278 I1 254 (50); sh 278 12 254(11.S);sh 278 20 13 254 (3.2); sh 278 14 254(I.16);sh 278 shoulder Fractions 7-10 are combined. This solution is mixed with 1 l. of acetone to give a precipitate; yield, 1.2 g. of clindamycin-3- 5 -guanylate) having the following structure:

CH3 R1 1 H-CCl C ONH-TII 3 (|)HSCHa V I CLDCIIzC I13 N N J H .1

2 N N 0 I1 0 I A:1"0CHz/ H OH Clindamycin-3-(5 '-guanylate) has the following chemical and physical properties:

Potentiometric Titration In water: pKa', 7.6

Eq. wt., 721

Specific Rotation [011 +69 (c, 1.0, water) Infrared Spectrum The infrared spectra in both mineral oil mull and KBr pellet are as follows: 5

IN MINERAL OIL MULL Band Band 1refro Intenquency Intenquency lnU-u- Band frequency (emr sity (cmr sity (cmsity 1,530 S 1 1,050 1, 457 S 1187 .\I 1,400 M 1 63 M 2 1.375 S 925 .\I 1,365 S 885 1,315 M 855 .\I I 1, 250 S 705 .\I 1,215 S 780 .\I 1 1,080 S 717 M 1,065 S 680 M In K131 P011013 1, 570 S 1, 005 S 1, 530 M 1, 045 S 1,450 M 1185 M 1,405 M .125 M 1, 380 M 885 M 1,355 M 855 M I 1, .255 M 800 M 1,030 S 1, 210 S 780 .\I 1,505 s 1,030 s 1 S11. 2 Oil.

U.V. Spectrum: In water at the folio wing pH's:

Max. 0 a pH 1.0 256 14.49 11,150 277 sh 9.69 7,456 pH 7.0 254 16.18 12,450 273 sh 11.21 8,626 pH 11.0 259 13.95 10,734 266 13.78 10,603

Reaction with Enzymes Crude Alkaline phosphatase Treatment with alkaline phosphatase yields clindamycin identified by thin-layer chromatography (silica gel, ethyl acetate-acetone-water (8:5: 1 v/v)). Venom Diesterase Treatment with venom diesterase yields clindamycin and guanosine-S '-phosphate.

In Vivo Activity Clindamycin-3-(5'-guanylate) does not possess antibacterial activity against Sarcina lutea in vitro. However, it is active in vivo (S.Q., mice, S. aureus) with a CD, of 26 mg./kg.

EXAMPLE 2 Upon substituting the microorganism Streptomyces venezuelae, NRRL 3527, for the microorganism S. coelicolor Miiller, NRRL 3532, in Example 1, there are obtained the clindamycin-3-nucleotides disclosed in Example 1.

EXAMPLE 3 Upon substituting lincomycin for clindamycin in the fermentation medium of Example I, there are obtained lincomycin-S-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 4 Upon substituting 1'-demethyl-c1indamycin for clindamycin in the fermentation medium of Example 1, there are obtained 1-demethyI-clindamycin-3-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 5 Upon substituting l-demethyI-4'-depropyl-4-pentylclindamycin for clindamycin in the fermentation medium of Example 1, there are obtained 1-demethyl-4-depropyI-4'-pentylclindamycin-3-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 6 Upon substituting 4'-depropyI-4'-ethyl lincomycin for clindamycin in the fermentation medium of Example 1, there are 17 obtained 4'-depropyl-4'-ethyl-lincomycin-S-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 7 Upon substituting l-demethyl-l -ethyl lincomycin for clindamycin in the fermentation medium of Example 1, there are obtained l'-demethyl-l '-ethyl lincomycin-3-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 8 Upon substituting l'-demethyl lincomycin for clindamycin in the fermentation medium in Example 1, there are obtained l-demethyl lincomycin-3-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

EXAMPLE 9 Upon substituting celesticetin for clindamycin in the fermentation medium in Example 1, there are obtained celesticetin-3-nucleotides wherein the nucleotide moieties are the same as disclosed in Example 1.

In the following examples, as above, the nucleotide moieties of the compounds of the examples are the same as disclosed in Example 1, Le, cytidylate, adenylate, uridylate and guanylate.

EXAMPLE l LlNCOMYClN-3-NUCLEOTIDE- AMMONIUM SALT A lincomycin-3-nucleotide in the zwitte'rionic form is dissolved in a minimum amount of water and diluted with an equal amount of ethanol. The solution is cooled in an icewater bath and then saturated with ammonia gas. The solution is taken to dryness at 30 C. under high vacuum. The residue is dissolved in a minimum amount of methanol and diluted with volumes of ether to precipitate lincomycin-3-nucleotide as the ammonium salt.

EXAMPLE l1 Aqueous Oral Drops A lincomycin-3-nucleotide I00 gm. Propyl paraben 0.25 gm. Methyl paraben 0.75 gm. Sorbic acid l.0 gm. Sodium hydroxide, 4 N aqueous q.s.

to pH 7.5 Water, deionized q.s. I000 ml.

EXAMPLE l2 SYRUP An aqueous oral preparation containing 400 mg. of a lincomycin-B-nucleotide in each 5 milliliters is prepared from the following ingredients:

A lincomycin-3-nucleotide 800 gm. Methyl paraben, U.S.P. 7.5 gm. Propyl paraben, U.S.P. 2.5 gm. Sorbic acid [0 gm. Saccharin sodium 6.5 gm. Glycerin 3000 ml. Tragacanth powder I00 gm. Orange oil flavor gm. F.D. and C. orange dye 7.5 gm. Sodium hydroxide, 4 N aqueous q.s. pH 7.5 Deionized water q.s. 10,000 ml.

in place of a lincomycin-3-nucleotide in Examples 11 and 12, there can be substituted a 7 (S)-chloro-7-deoxylincomycin-S-nucleotide, as well as the water soluble salts of a 7(S)-chloro-7-deoxylincomycin-3-nucleotide, for example, the alkali metal salts including the ammonium salt.

The aqueous formulations of Examples 11 and 12 are particularly useful as pediatric preparations and can be administered orally in the same dosages as lincomycin.

EXAMPLE l3 CAPSULES One thousand two-piece hard gelatin capsules for oral use, each containing 350 mg. of a l'-demethylclindamycin-3- nucleotide are prepared from the following types and amounts of materials:

A l'-demethylclindamycin-3-nucleotide 350 gm. Corn starch 50 gm. Talc 25 gm. Magnesium stearate 2.5 gm.

The materials are thoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the systemic treatment of infection in adult humans by the oral administration of one capsule every 4 hours.

Using the procedure'above, capsules are similarly prepared with a l'-demethylclindamycin-B-nucleotide in 50, I25, 250 and 500 mg. amounts by substituting 50, 125, 250 and 500 gm. of a l'-demethylclindamycin-3-nucleotide for the 350 gm. used above.

EXAMPLE l4 TABLETS One thousand tablets for oral use, each containing 500 mg. of a 1-demethyl-4'-depropyl-4'-pentyl-clindamycin-3-nucleotide are prepared from the following types and amounts of materials:

A l'-demethyl-4'-depropyl-4- pentylclindamycin-3-nucleotide 500 gm. Lactose 50 gm. Corn starch 65 gm. Magnesium stearate 3 gm. Light liquid petrolatum 3 gm.

EXAMPLE [5 PARENTERAL PREPARATION A sterile aqueous preparation for intramuscular use, containing in 1 ml. 300 mg. of a celesticetin-3-nucleotide is prepared from the following types and amounts of materials:

A celesticetin-3-nucleotide 300 gm. Benzyl alcohol 9 gm. Water for injection, q.s. I000 ml.

The sterile drug is dispensed in the sterile benzyl alcoholwater vehicle and filled into vials and the vials sealed.

EXAMPLE l6 ANIMAL FEED One thousand gm. of a feed mix is prepared from the following types and amounts of ingredients:

A 4-depropyl-4'-ethyl lincomycin 3-nucleotide 20 gm. Soybean meal 390 gm. Fish meal 400 gm. Wheat germ oil 50 gm. Sorghum molasses gm.

The ingredients are mixed together and pressed into pellets. The composition can be fed to laboratory animals, i.e., rats, mice, guinea pigs, and rabbits for prophylaxis during shipping.

For larger animals the composition can be added to the animals regular feed in an amount calculated to give the desired dose of active material.

EXAMPLE 17 PARENTERAL PREPARATION A lincomycin-3-nucleotide 300 gm. Benzyl alcohol 9 gm. Water for injection, q.s. 1000 ml.

The sterile drug is dispensed in the sterile benzyl alcoholwater vehicle and filled into vials and the vials sealed.

EXAMPLE l8 7-DEOXY-7(S)- METHOXYLINCOMYCIN HYDROCHLORIDE Part 18-A:

Methyl nide N-acetyl-7-deoxy-7(S )-methoxy-a-thiolincosami- OMe AcNH- HO P (on l I SMe OH VII A suspension of 2.35 gms. of methyl 6,7-aziridino-6- deamino-7-deoxy-a-thiolincosaminide (VI) was maintained with stirring in 25 ccs. of methanol. To the suspension was then added 2.04 gms. of acetic anhydride. After stirring at room temperature for 1 hour the solvent was removed on a rotary evaporator at 40 C./7 mm. The resulting solids were then chromatographed on a 4.8 X 94 cm. column of silica gel using 1 MeOH: 10 CHCl as the solvent system. The weight ofthe silica was 750 gms. After a forerun of 1,000 ml., 50 ml. fractions were collected. Fractions 31-85 were combined, and evaporated to dryness yielding 3.2 gms. of methyl N-acetyl- 7(S)-methoxy-7-deoxy-a-thiolincosaminide (Vll) as a colorless amorphous solid, having the molecular weight by mass spectrometry of 309, compared with the calculated molecular weight of 309.38.

The starting aziridino compounds of Fonnula V1 can be obtained by dehydrohalogenation of methyl 7(S)-chloro-7- deoxy-a-thiolincosaminide (Belgian Pat. No. 705,427, Apr. 22, 1968; U.S. application Ser. No. 692,727, filed Dec. 22, 1967). The dehydrohalogenation is effected with anhydrous sodium carbonate by heating at reflux in dimethylformamide (Belgian Pat. 732,352, Oct., 1969; U.S. application Ser. No. 725,531, filed Apr. 30, 1968).

Part 18-B:

Methyl 7-deoxy-7(S)-methoxy-a-thiolincosaminide (V111) (Methyl 6,8-dideoxy-7-O-methyl-6-amino-1-thio-L-threo-a- D-galacto-octopyranoside) (l I [a NH2 110 l OH l s Me 0 H VIII A solution of 3.2 gms. of methyl 7-deoxy-7(S)-methoxy-athiolincosaminide (V11) in 25 gms. of hydrazine hydrate was heated under gentle reflux with stirring in an oil-bath at 145 C. overnight. The solvent was removed from the colorless solution as completely as possible by distillation from an oilbath at 100 C./15 mm. and finally at high vacuum to give methyl 7-deoxy-7(S)-methoxy-a-thiolincosaminide as a colorless syrup. The syrup was chromatographed on 750 gms. of silica gel in a 4.8 X 97 cm. column using 1 MeOH:10 CHCl as the solvent system. After 1.4 liter forerun, 50 ml. fractions were collected. Fractions 281-600 were pooled and evaporated to dryness yielding 2.06 gms. methyl 7-deoxy- 7(S)-methoxy-a-thiolincosaminide (VIII) which on crystallization from acetonitrile yielded colorless needles having the following characteristics:

m.p.154-155C.

Anal. Calcd. for C H O NS:

C,44.92; H, 7.92; N, 5.24;S, 12.00;OMe, 11.61.

Found:

C, 45.20; H, 7.96; N, 5.08; S, 12.19; OMe, l 1.86.

M01. Wt. calcd.:

Found (mass spec.):

267 Part 18-C:

7-Deoxy-7(S)-methoxylincomycin hydrochloride OMe Pr =n-propyl O H To a suspension of 2.7 gms. of trans-1-methyl-4-propyl-L-2- pyrrolidinecarboxylic acid hydrochloride in 90 ccs. acetonitrile was added with stirring 2.89 gms. of triethylamine. The stirring was continued until all of the solid had dissolved; the reaction mixture was then cooled in an ice/methanol bath to 5 C., when a precipitate of triethylamine hydrochloride appeared. There was then added 1.78 gms. of isobutyl chloroformate dropwise keeping the temperature of the reaction at -5 to 3 C. Additional triethylamine hydrochloride precipitated and stirring was continued at 5 C. for 20 minutes. To the resulting reaction mixture was added 1.74 gms. of methyl 7- deoxy-7(S)-methoxy-a-thiolincosaminide (Vlll), dissolved in 10 ccs. of water. As the solids dissolved, the temperature rose to about 0 C. and stirring was continued for 2 hours, without further icing the cooling bath. The solvent was then removed on a rotary evaporator at 40 C./15 mm. to a brown viscous residue. This was dissolved in dilute hydrochloric acid and the solution (pH 2) extracted twice with chloroform and the combined extracts washed once with water. The aqueous phase containing the wash water was adjusted to pH 11 with sodium hydroxide (50 percent aqueous solution), saturated with sodium chloride and extracted three times with chloroform. The combined chloroform extracts were dried over anhydrous sodium sulfate and taken to dryness yielding 1.76 gms. of a tan amorphous solid. The tan amorphous solid was chromatographed on 750 gms. of silica gel in a 4.8 X 94 cm. column using 1 MeOH:15 CHCl as the solvent system. After 1.3 liters of forerun, 50 ml. fractions were collected. Fractions 60 through were pooled and taken to dryness yielding 7-deoxy-7(S)- methoxylincomycin free base as an almost colorless syrup. This free base was taken up in dilute aqueous HCl and the resulting solution filtered and freeze-dried yielding 801.4 mg. of 7-deoxy-7(S )-methoxylincomycin hydrochloride as a colorless amorphous solid having the following characteristics:

Anal. Calcd. for C,-,H,,O,N,S-HC1:

C, 49.93; H, 8.16;N, 6.13; S, 7.02.

Found (corrected for 5.14 percent H,O):

C, 49.44; H, 7.99; N, 6.20; S, 6.48.

M01. Wt. Calcd. for anhydrous free base:

Found (Mass spec.

EXAMPLE l9 ALTERNATIVE METHOD FOR PRODUCING METHYL 7-DEOXY-7(S)-METHOXY-a- THIOLINCOSAMINIDE (VIII) Part 1 9-A:

Methyl N-acetyl-6,7-aziridino-6-deamino-7-deoxy-2,3,4-tri- O-acetyl-a-thiolincosam inide (X) To a solution of 2.0 gms. of methyl 6,7-aziridino-6- deamino-7-deoxy-a thiolincosaminide (VI) in 20 ccs. of pyridine was added with stirring 10 ccs. of acetic anhydride and the reaction mixture left overnight at room temperature. The volatile material was removed as completely as possible from the reaction mixture on a rotary evaporator at 40 C./7mm., finally at high vacuum, to a colorless solid. The resulting solid was dissolved in chloroform, stirred with aqueous cadmium chloride to remove the pyridine, filtered and the chloroform layer washed twice with water, and dried over anhydrous sodium sulfate. On removal of the solvent on the rotary evaporator at 40 C./7 mm. methyl N-acetyl-6,7- aziridino-6-deamino-7-deoxy-2,3,4-tri-O-acetyl-a-thiolincosaminide (X) was obtained as a colorless crystalline solid, weight 3.1 gms. Recrystallization from ethyl acetate-Skellysolve B (technical hexane) gave colorless prismatic needles having the following characteristics:

[a],,+222(c,0.912, Cl-lCl Anal. Calcd. for c,,H,,o,Ns:

C, 50.61; H, 6.25; N, 3.47; S, 7.95.

Found:

C, 50.43; H, 6.33; N, 3.41;S, 8.31.

M01. Wt. calcd.:

Found (Mass spec.):

403 Part 19-B:

Methyl N-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7(S)-methoxytit-thiolincosaminide (XI) A mixture of 5 gms. of methyl N-acetyl-2,3,4-O triacetyl- 6,7-aziridino-6-deamino-7-deoxy-a thiolincosaminide (X), 50 cos methanol, and 5 ccs. glacial acetic acid was heated under gentle reflux in an oil bath at 130 C. for 6 hours. The solvent was removed from the colorless solution at 40 C./7 mm. on a rotary evaporator yielding a pale yellow syrup which crystallized. The crystals were taken up in methylene chloride solution, washed with saturated aqueous sodium bicarbonate, then with water, and then dried over anhydrous sodium sulfate. Removal of the solvent as above gave methyl N-acetyl-2,3,4- tri-O-acety1-7(S)-methoxy-7-deoxy-a-thiolincosaminide (XI) as colorless crystals (5.31 gms.). Crystallization from ethyl acetate-Skellysolve B gave fine colorless needles having the following properties:

m.p. 235-236C.

[01],, +205 (c, 0.9952, CHCl Anal. Calcd. for C l-I,,O,NS:

C, 49.64; H, 6.71; N, 3.22; s, 7.36; OMe, 7.13.

Found:

C, 49.77; H, 6.92; N, 3.65; S, 7.90; OMe, 7.38.

Mol. Wt. calcd.:

Found (Mass spec.):

On hydrazinolysis by the procedure of Part 18-B there is obtained methyl 7-deoxy-7(S)-methoxy-a-thiolincosaminide (VIII).

'EXAMPLEZO- MODIFICATION OF EXAMPLE 18 Part 20-A:

Methyl N acety1-2,3,4-tri-O-acetyl-7-deoxy-7(S)-methoxya-thiolincosaminide (XI) and Methyl N-acetyl-2,3,-di-O- acetyl-7-deoxy-7(S)-methoxy-a-thiolincosaminide (XII) oMe OMe AcNH- ACNH...

o' o AcO HO l I A l SMe I l) Mo Ac Ac XI XII To 26.61 gms. of methyl N-acetyl-7-deoxy-7(S)-methoxy-athiolincosaminide (VII) in cos. of pyridine there was added 50 ccs. of acetic anhydride with stirring and the reaction mixture allowed to stand overnight at room temperature. The volatile materials were then removed by distillation on a rotary evaporator at 40 C./7 mm. and finally under high vacuum. The residue was dissolved in chloroform and washed with saturated aqueous sodium bicarbonate. The aqueous layer was washed with chloroform and the combined chloroform extracts stirred with aqueous cadmium chloride to remove the pyridine.

The precipitate was filtered off and washed well with chloroform and the chloroform layer separated, washed twice with water and dried over anhydrous sodium sulfate. On removal of the solvent on a rotary evaporator at 40 C./7 mm. a pale yellow syrup which crystallized on standing was obtained. On recrystallization from ethyl acetate-Skellysolve B, the produce was obtained as small colorless, flattened needles, and had the following characteristics:

m.p. 245-247C.

[01],, +202 (c, 0.7142, CI-ICI Anal. Calcd. for C H O NS:

C, 49.64; H, 6.71; N, 3.22; S, 7.36; OMe, 7.13.

Found:

C, 49.24; H, 6.75; N, 3.34; S, 7.52; OMe, 7.17.

M01. Wt. calcd.:

Found (Mass spec.):

The above material by Craig countercurrent distribution using as a solvent system 1 EtOH:1 H O:1 EtOAc:1 cyclohexane 'was shown to contain 70 percent of methyl N-acetyl- 2,3,4-tri-O-acetyl-7-deoxy-7(S)-methoxy-a-thiolincosaminide (XI) and 30 percent of methyl N-acetyl-2,3-di-O-acetyl-7- deoxy-7(S)-methoxy-a-thiolincosaminide (XII). After 500 transfers, fractions from tubes 225-310 were pooled (K value 1.14) and evaporated to dryness and on recrystallization from ethyl acetate-Skellysolve B gave methyl N-acetyl-2,3,4-tri-O- acetyl-7-deoxy-7(S)-methoxy-a-thiolincosaminide (XI) as fine colorless needles, identical with the product of Part 19-B.

Fractions from tubes -220 (K value 0.59) were pooled and evaporated to dryness and on recrystallization from ethyl acetate-Skellysolve B gave methyl N-acetyl-2,3-di-O-acetyl-7- deoxy-7(S)-methoxy-a-thiolincosaminide (XII) as colorless chunky needles having the following characteristics:

m.p. 189-190 C.

[01],, +275 (0, 1.0188, Cl-lCl Anal. Calcd. for C,,I-I,-,O NS:

C, 48.84; H, 6.92; N, 3.56; S, 8.15; OMe, 7.89.

Found:

C, 48.71; H, 7.1 l; N, 3.93; S, 7.96; OMe, 7.98.

M01. Wt. calcd.:

Found (Mass spec.):

393 Part 20-B:

Acetylation of methyl N-acetyl 2,3-di-O-acetyl-7-deoxy-7 (S)-methoxy-athiolincosaminide (XII) To a solution of 200 mg. of methyl N-acetyl-2,3-di-O- acetyl-7-deoxy-7(S)-methoxy-a-thiolincosaminide (XII) in 20 cos. of pyridine was added ccs. of acetic anhydride with stirring and the reaction mixture left at room temperature overnight. The solvent was removed from the colorless reaction solution on a rotating evaporator at 40 C./7 mm. finally at 40 C./high vacuum. The syrupy residue was dissolved in chloroform, washed with dilute aqueous HCl (one-half normal), twice with water, with saturated sodium bicarbonate solution and twice with water, and dried over anhydrous sodium sulfate. The solvent was then removed on a rotating evaporator at 40 C./7 mm. yielding methyl N-acetyl-2,3,4-tri- O acetyl-7-deoxy-7(S)-methoxy-a-thiolincosaminide (XI) as a colorless syrup which crystallized on standing.

On hydrazinolysis of the products of Part -A and 20B, there is obtained methyl 7-deoxy-7(S)-methoxy-a-thiolincosaminide (VIII).

EXAMPLE 21 ALTERNATIVE FOR EXAMPLE 18 Part2l-A:

Methyl N-acetyl 6,7-aziridino-6-deamino-7-deoxy-athiolincosaminide (XIII) CH AcN HO O so. l SMe 01-1 XIII To a suspension of 2.3 gms. of methyl 6,7-azlridino-6- deamino-7-deoxy-a-thiolincosaminide (VI) in ccs. isopropyl alcohol, there was added with stirring 2.04 gms. acetic anhydride. Most of the solid appeared to go into solution to be replaced by new solid. The reaction mixture was stirred overnight at room temperature, then filtered and the residue washed with isopropyl alcohol and dried in a vacuum oven at 60 C./l5 mm. There was obtained 2.28 gms. of methyl N-acetyl-6,7-aziridino-6-deamino-7-deoxy-a-thiolincosaminide as colorless platelets having the following properties:

m.p. 145C.

Ana]. Calcd. for C,,H,,,O NS:

C,47.63; H, 6.91; N,5.05;S, 11.56.

Found:

C,47.57; H, 6.71; N, 5.23;S, 11.29.

Mol. Wt. calcd.:

Found (Mass spec.):

277 Part 2 l-B:

Methyl nide (VII) On treating methyl N-acetyl 6,7-aziridino6-deamino-7- deoxy-a-thiolincosaminide (XIII) with methanol and acetic acid under reflux, there is obtained methyl N-acetyl-7-deoxy- 7(S)-methoxy-a-thiolincosaminide (VII) identical with the product of Part 18-A.

N-acetyl-7-deoxy-7( S )-methoxy-a-thiolincosami- EXAMPLE 22 7-DEOXY-7(S)-ETHOXYLINCOMYCIN HYDROCHLORIDE Part 22-A:

Methyl N-acetyl-7-deoxy-7(S)-ethoxy-a-thiolincosaminide (XIV) OElG ACNH K E PLM.

OH XIV CH CH3 OEl; -OEt AcNH- AcNH- HO l Ae [{OAc i l l Mo Me 0110 OAc XV XVI 0n treating the methyl N-acetyl-7-deoxy-7(S)-ethoxy-athiolincosaminide (XIV) with acetic anhydride and pyridine by the process of Part 20-A there is obtained methyl N-acetyl- 2,3,4-tri-O-acetyl-7-deoxy-7(S)-O-ethyl-a-thiolincosaminide (XV) together with a minor amount of N-acetyl-2,3-di-O- acetyl-7-deoxy-7(S)-ethoxy-a-thiolincosaminide (XVI). The products (isolated on a Craig in 500 transfers using ethanolzwaterzethyl acetate:cyclohexane (1:1:111) as the solvent system) are characterized as follows: Mixture:

[11],, +247 (c, 0.665, CHCI Anal. Calcd. for C H O NS:

C, 50.76; H, 6.95; N, 3.12; S, 7.13; OEt, 10.02.

Found:

C, 50.42; H, 7.07; N, 3.18; S, 7.37; OEt, 11.85. Pure XV (K= 1.59): m.p. 254255 C.

[01],, +199 (0, 0.8638, CHCI Anal. Calcd. for C H O NS:

C, 50.76; H, 6.95; N. 3.12; S, 7.13;OEt,10.02.

Found:

C, 50.75; H, 7.06; N, 3.37; S, 7.31;0Et, 10.25.

Mol. Wt. calcd.:

Found (Mass spec.):

449 Pure XVI (K 0.87):

m.p. 215.5-216.5 C.

[01],, +26l (0, 1.0448, CHCI,)

Anal. Calcd. for C H O NS:

C, 50.11;H, 7.17; N, 3.44; S, 7.87.

Found:

C, 50.17, H, 7.30; N, 3.50; S, 7.62.

M01. Wt. calcd.:

Found (Mass spec.):

407 Part 22-C:

Methyl 7-deoxy-7(S)-ethoxy-a-thiolincosaminide (XVII) XVII On subjecting the products of Part 22-B, that is, the mix- 10 ture, the pure XV or the pure XVI to hydrazinolysis, there is obtained methyl 7-de0xy-7(S)-ethoxy-a-thiolincosaminide (XVII) having the following characteristics:

m.p. 194196 C. [01],, +252 (0, 0.7438, 11,0)

Anal. Calcd. for C ,I-I,,O,NS:

C, 46.95; H, 8.24; N, 4.98; S, 1 1.40.

Found:

C, 46.66; H, 8.09; N, 5.26; S, 11.33.

Mol. Wt. calcd.:

Found (Mass spec.):

28 1 Part 22-0:

7-Deoxy-7(S)-ethoxylincomycin hydrochloride (XVIII) Me O l I H:

( no 'HCI Me=metl1yl i OII ET=cthyl Pr=propyl OH XVIII Following the procedure of Part 18-C, methyl 7-deoxy- 7(S)-ethoxy-athiolincosaminide (XVII) is converted to 7- deoxy-7(S)-ethoxylincomycin hydrochloride having the following characteristics:

m.p. colorless amorphous solid [a D +109 (0, 0.9824, H O) Anal. Calcd. for C ,,H O,N,S-I-ICl:

C, 50.99; H, 8.35; N, 5.95; CI, 7.53; S, 6.81; OEt, 9.57.

Found: (corrected for 5.07 percent water) C, 50.54; H, 8.19; N, 5.63;Cl, 7.61; S, 6.95; OEt, 10.16.

EXAMPLE 23 Part 23A:

Methyl N-acetyl-7-deoxy-7(S)-propoxy-a-thiolincosaminide (XIX), methyl N-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7(S)- propoxya-thiolincosaminide (XX), and methyl N-acetyl-7- deoxy-7(S)-propoxy-2,3-di-O-acetyl-a-thiolincosaminide On treating the methyl N-acetyl-6,7-aziridino-6-deamino-7- deoxy-a-thiolincosaminide (XIII) with propanol and acetic acid under gentle reflux, there is obtained methyl N-acetyI-7- deoxy-7(S)-propoxy-a-thiolincosaminide (XIX) from which on acetylation with acetic anhydride in pyridine by the procedure of Part 22-8, there is obtained methyl N-acetyl- 26 2,3 ,4-tri-O-acetyl-7(S)-propoxy-7deoxy-a-thiolincosaminide (XX) containing a rninor'amount of methyl N-acetyI-2,3-di-0- acetyl-7(S)-propoxy-7-deoxy athiolincosaminide (XXI) having-the following characteristics: Mixture:

m.p. 240242 C. [01],, +207 (0, 0.9054, CHCI,) Anal. Calcd. for C, I'I,,O,',NS: C, 51.81, H, 7.17; N, 3.03; S, 6.92. Found: C, 51.41; H, 7.33; N, 3.16; S, 6.92. M01. Wt. calcd.: 463.60 Found (Mass spec.): 463 Pure XX:

m.p. 241.5242.5 C. [a],,+193 (0, 0.9254 CllCl Anal. Calcd. for C I-I, ,O,NS: C, 51.81; H, 7.17; N, 3.03; S, 6.92. Found: C, 51.77; H, 7.02; N, 3.37; S, 6.84. M01. Wt. calcd.: 463.60 Found (Mass spec.): 463

Part 23-B:

Methyl 7-deoxy-7(S)-propoxy-a-thiolincosaminide (XXII) NHz- H0 F l S Me H XXII On hydrazinolysis of the above products )Iart 23-A) there is obtained methyl 7-deoxy-7(S)-propoxy-a-thiolincosaminide (XXII). Part 23-C:

7-Deoxy-7(S)-propoxylincomycin hydrochloride(XXIII) 1 OH I l SMe On acylation with trans-l-methyl-4-propyl-L-2-pyrrolidinecarboxylic acid by the procedure of Part l8-C, there is obtained 7-deoxy-7(S)-propoxylincomycin hydrochloride (XX- III).

EXAMPLE 24 -HCl Part 24A:

Methyl N-acetyl-Z,3,4-tri-O-acetyl-7-deoxy-7(S)- isopropoxy-a-thiolincosaminide (XXIV) On hydrazinolysis of compound XXIV (Part 24-A), there is obtained methyl 7-deoxy-7(S)-isopropoxy-a-thiolincosaminide having the following characteristics:

m.p. 21 3 C.

[01],, +225 (c, 0.376, H O) Anal. Calcd. for C,,H, O NS:

C, 48.79; H, 8.53; N, 4.74; S, 10.86.

Found:

C, 48.52; H, 8.55; N, 5,26;S, 10.84.

Mol. Wt. Calcd.

Found (Mass spec.)

295 Part 24C:

7-Deoxy-7(S)-isopropoxylincomycin hydrochloride (XX- VI) ill/Io N XXVI Following the procedure of Part l8-C, compound XXVI (Part 24-C) is converted to 7-deoxy-7(S)-isopropoxylincomycin hydrochloride having the following characteristics:

m.p. amorphous Anal. Calcd. for C,,H O,N,S-HCI:

C, 51.99; H, 8.52; N, 5.78; S, 6.61;Cl, 7.31.

Found (corrected for 4.36 percent H,O):

C, 51.72; H, 8.33; N, 5.59; S, 6.35; CI, 7.29.

Mol. wt. calcd. (free base):

Found:

Activity:

about the same as lincomycin EXAMPLE 25 (III:

Part 25-A:

Methyl N-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7(S)-cyclohexyloxy-a-thiolincosaminide (XXVII) O Cyclohexyl AeNII- AcO A0 XXVII LII Following the procedure of Part 19-8 substituting the methanol by cyclohexanol, there is obtained methyl N-acetyl- 2,3.4-tri-O-acetyl-7-deoxy-7(S)-cyclohexyloxy-a-thiolincosaminide (XXVII) having the following characteristics:

Anal. Calcd. for C,,H,-,O,NS:

C, 54.85; H, 7.41; N, 2.78; S, 6.37.

Found:

C, 54.93; H, 7.53; N, 2.87; S, 6.65.

Mol. Wt. Calcd.

Found (Mass spec.):

503 Part 25-B:

Methyl (XXVIII) 7( S)-cyclohexyloxy-7-deoxy-a-thiolincosaminide 0 Cycle hexyl On hydrazinolysis of compound XXVII (Part 25-A), there is obtained methyl 7(S)-cyclohexyloxy-7-deoxy-a-thiolincosaminide (XXVIII) Part 25-C:

7(S)-cyclohexyl-7-deoxylincomycin hydrochloride XXVIII Following the procedure of Part l9-C, methyl 7(8)- cyclohexyloxy-7-deoxy-a-thiolincosaminide (XXVIII) is converted to 7( S)-cyclohexyloxy-7-deoxylincomycin hydrochloride.

EXAMPLE 26 Part 26-Az Methyl N-acetyl-7-deoxy-7(S)-2-hydroxyethoxy-a-thiolincosaminide (XXIX) and methyl N-acetyl-2,3,4-tri-O-acetyl- 7(S)-2'-acetoxyethoxy-7-deoxy-a-thiolincosaminide (XXX) on. on.

- 0CH2CII20II oorncmosc AcNII- A N no 0 AcO SM: SMe 0H OAc XXIX xxx Following the procedure of Part l8-A substituting the methanol by 2-hydroxy-ethanol, there is obtained methyl N- acetyl-7-deoxy-7(S)-2'-hydroxyethoxy-a-thiolincosaminide (XXIX) which when acylated by the procedure of Part 20-A but with heating on a steam bath to produce the fully acylated product gives methyl N-acetyl-2,3,4-tri-O-acetyl-7(S)-2'- acetoxyethoxy-7-deoxy-a-thiolincosaminide having the following characteristics:

[a], +1 72 (c, 1.010, CHCI,)

Anal. Calcd. for C H O NS: C,49.69; H, 6.55; N, 2.76; S, 6.32. Found: C, 49.56; H, 6.63; N, 2.90; S, 6.63. M01. Wt. calcd.: 507.55 Found: 507 Part 26-B2 Methyl nide (XXXI) SMo l LII XXXI On hydrazinolysis of methyl N-acetyl-Z,3,4-tri-O-acetyl-7- deoxy-7(S)-2'-acetoxyethoxy-a-thiolincosaminide (XXXI), there is obtained methyl 7-deoxy-7(S)-2'-hydroxyethoxy-athiolincosaminide having the following characteristics:

m.p. 178.5-179.5C.

[a],,+243 (c, 0.662, H O) Anal. Calcd. for C H O NS:

C,44.43; H, 7.80; S, 10.78,N, 4.71.

Found:

C, 44.40; H, 7.99; S, 10.51 N, 4.60.

M01. Wt. calcd.:

Found (Mass spec.):

Part 26-C:

Methyl 7-deoxy-7(S)-2'-hydroxyethoxylincomycin hydrochloride (XXXII) (Ill;

'llCl XXXII Following the procedure of Part 18-C, methyl 7-deoxy- 7(S)-2' hydroxyethoxy-a-thiolincosaminide (XXXI) is convened to 7-deoxy-7(S)-2'-hydroxyethoxylincomycin hydrochloride having the following characteristics:

m.p. amorphous [01],, 105 (c, 1.102, H O) Anal. Calcd. for C, H,,0-,N,S-HCl:

C, 49.32; H, 8.07; N, 5.75; S, 6.58;Cl, 7.28.

Found: (corrected for 2.1 1 percent H,O)

C, 49.61; H, 7.85; N, 5.54; S, 6.46; CI, 7.26.

M01. wt. calcd. (free base):

Found (mass spec.):

Activity:

about one-third lincomycin EXAMPLE 27 Part 27-A:

Methyl N-acetyl-7-deoxy-7(S)-2'-methoxyethoxy-a-thiolincosaminide (XXXlll) and methyl N-acetyl-2,3,4-tri-0-acetyl- 7-deoxy-7(S)-2'-methoxyethoxy-a-triolincosaminide (XXX- IV) 7-deoxy-7(S)-2'-hydroxyethoxy-a-thiolincosamil0 C H3 C Ha -o c1120 1120M; -o c memoirs AeNH- AcNH- AcO OM0 l and l OMe (L SMe SMe Me OMe XXXIII xxxrv Following the procedure of Part 18-A but substituting the methanol by Z-methoxyethanol, there is obtained methyl N- acetyl-7-deoxy-7(S)-2'-methoxyethoxy-a-thiolincosaminide (XXXlll) which on acetylation by the procedure of Part 20-A but with heating on a steam bath to produce the fully acetylated product yields methyl N-acetyl-2,3,4-tri-O-acetyl-7- deoxy-7(S)-2'-methoxyethoxy-a-thiolincosaminide (XXXIV) which is characterized as follows:

m.p. 222223 C. [11],, +177 (c, 1.079, CHCl,) Anal. Calcd. for C H O NS: C, 50.09; H, 6.94; N, 2.92; S, 6.69; OMe, 6.47. Found: C, 50.13; H, 7.00; N, 2.77; S, 6.33; OMe, 7.28. M01. Wt. calcd.: 479.54 Found (Mass spec.): 479

Part 27-B:

Methyl 7-deoxy-7(S)-2'-methoxyethyl-a-thiolincosaminide (XXXV) -OCHCH20M8 NHz- I OH /I I l/ S Ma H XXXV Me I 111 --0c1I20moMe l l CNH-- I SMe lH XXXVI Following the procedure of Part l8-C, methyl 7-deoxy- 7(S)-2-methoxyethoxy-a-thiolincosaminide (XXXV) is converted to 7-deoxy-7(S)-2'-methoxyethoxylincomycin hydrochloride having the following characteristics:

m.p. amorphous [a], +87 (c, 0.575, H,O)

Anal. Calcd. for C,,H,,O,N,S'HCI:

C, 50.33; H, 8.25; N, 5.59; S, 6.40; Cl, 7.08.

Found:'(corrected for 4.17 percent H,O)

-IIC1 Thiolincosaminide (XXXVII) (Methyl 6-Amino-6,8-Dideoxy- L-Threoa-D-Galacto-0ctopyranoside) nide (XXXVIll) (methyl 6-acetamido-6,8-dideoxy-L-threo-a- D-galacto-octopyranoside) xxxvrn 3 5 To a solution of 2.35 gms. of methyl 6,7-aziridino-6- deamino-7-deoxy-a-thiolincosaminide (VI) in 25 ccs. of water was added 2.04 gms. of acetic anhydride and the solution left at room temperature overnight. The solution was then taken to dryness on a rotary evaporator at 40 C/7 mm. to give a colorless syrup which was chromatographed on 750 gms. of silica gel in a 4.8 X98 cm. column using 1 MeOH:7 CHCl, as the solvent system. After a forerun of 550 ml., 50 ml. fractions were collected. Fractions 90 through 160 were pooled and taken to dryness to give 2.3 gins. of methyl N-acetyl 7-deoxy- 7(S)-hydroxy-a-thiolincosaminide as a colorless solid which crystallized from methanol as colorless rods having the following characteristics:

Anal. Calcd. for C,,H,,O,NS:

C, 44.73; H, 7.17; N, 4.74;S, 10.86.

Found:

C, 44.89; H, 7.02; N, 5. 16; S, 10.64.

Mol. Wt. calcd.:

Found (Mass spec.):

295 Part 28-8:

Deacetylation The crystallized material from Part 28-A was combined with the mother liquors and taken to dryness on a rotary evaporator at 40 C./7 mm. to give 2.01 grns. solid which was heated overnight under gentle reflux with 40 ccs. of hydrazine 65 hydrate with stirring. The solvent was removed from the colorless solution on a rotary evaporator at 7 mm. pressure in an oil bath at 120 C. The resulting colorless crystalline residue on recrystallization from methanol gave methyl 7-deoxy-7(S)- hydroxy-a-thiolincosaminide (XXXVll) as colorless platelets 70 having the following characteristics:

[a], +280 (c, 0.7728, H,O)

Anal. Calcd. for C,H,.O NS:

C, 42.67; H, 7.56; N, 5.53; S, 12.66.

Found: C, 42.81; H, 7.69;N, 5,85; S, 12.73. Mol. Wt. calcd.: 253.32 Found (Mass spec.): 253

EXAMPLE 29 Methyl N-Acetyl-2,3,4-Tri-O-Acetyl-7(S)- ethoxy-7-Deoxy-a-Thiolincosaminide (XV) and Methyl N- Acetyl-2,3,4-Tri-O-Acetyl-7(S)-Acetoxy-7-Deoxy-a- Thiolincosaminide (XXXlX) CH1 CH1 -o m -0Ac AcNH- AeNH- A00 AcO I 01m Bud I 0A0 l Me Me 0A0 Ac xv xxxIx Following the procedure of Part 19-B but substituting the methanol by ethanol, there is obtained methyl N-acetyl-2,3,4- tri-O-acetyl-7(S)-ethoxy-7-deoxy -a-thiolincosaminide (XV) identical with the product of Part 22-5 and a minor amount of N-acetyl2,3,4-tri-O-acetyl-7(S)-acetoxy-7-deoxy-a-thiolincosaminide (XXXIX) which can be separated by Craig countercurrent distribution using 1 EtOH:1 H,O:l EtOAc:l.5 cyclohexane as the solvent system in 500 transfers. The minor component (XXXlX) was obtained from tubes numbers -200 (K 0.52), and the major component (XV), from tubes numbers 260-330 (K 1.43). The minor component (XXXIX) crystallized from ethyl acetate as colorless needles having the following characteristics:

[01],, +182 (c, 0.5898, CHCl,)

Anal. Calcd. for C,,H,,O. NS:

C, 49.22; H, 6.31; N, 3.02; S, 6.92.

Found:

C, 49.17; H, 6.51; N, 3.08; S, 6,81.

Mol. Wt. calcd.:

Found (Mass spec.):

On subjecting the minor component to hydrazinolysis there is obtained methyl 7-deoxy-7(S)-hydroxy-a-thiolincosaminide (XXXVll) identical with the product of Part 28-8.

Example 30 Part 30-A:

2-Hydroxyethyl N-acetyl-2,2,3 ,4-tetra-O-acetyl-7-O- methyl l-thio-a-lincosaminide (XL) CHI CHgO- AeNH'-- 2'-Hydroxyethyl l-thio-a-celestosaminide (1.0 gm.) (Example 3 of U.S. Pat. No. 3,255,174) was left overnight in solution in pyridine (25 ccs.) and acetic anhydride (l2 ccs.) Removal of the solvent in vacuo gave a colorless oil which was dissolved in chloroform, 'washed with water, dilute aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate, water and dried over anhydrous sodium sulfate. Solvent removal in vacuo gave a syrup (2.03 gms.) which on crystallization from ethyl acetate-Skellysolve B yielded 2" hydroxyethyl N-acetyl-Z' ,2,3,4-tetra-O-acetyl-7-0-methyll thio-a-lincosaminide (Formula XL) in squat, colorless prisms,

m.p. 143-144 C. Skellysolve B is a brand of technical hexane.

Anal. Calcd. for C H O NS:

C, 49.68; H, 6.54; N, 2.76; S, 6.32 percent.

Found:

C, 49.66; H, 6.50; N, 2.91 S, 6.34 percent.

[a],,+2l6 (0, 0.7746, CHCI Part 30-8:

Methyl N-acetyl-2,3,4-tri--acetyl-7-O-methyl-1-thio-aand -fi-lincosaminides XL] and XLll) cm om omoemo- AcNH-- AcNH ls A00 A00 s CH:

(0A0 I and I 0.40 I s cm 0A0 0A0 XLI XLII A solution of 5.05 gms. (1.62 ccs) of bromine in 100 ccs. of chloroform was added over approximately 30minutes from a pressure-equalized dropping funnel under anhydrous conditions to a stirred solution of 10 gms. of 2'-hydroxyethyl N- acetyl-2',2,3,4-tetra-O-acetyl-1-thio-a-celestosaminide prepared by the procedure of Part 30A in 200 ccs. of chloroform. Initially, the bromine color disappeared immediately; later, a deep orange-red color developed. After stirring for an additional 30 minutes at room temperature, solvent was removed on a rotating evaporator at 40 C./7 mm., giving a yellow-orange syrupy residue. This was redissolved in chloroform, the solvent removed in vacuo, and the process repeated till the distillate became colorless, leaving a yellowish amorphous residue of 1-bromo-7-O-methyl-a-lincosamine tetraacetate of the formula CHa0 AcNH- XLIII The residue was dissolved in 200 ccs. of dry dimethylformamide, 4.5 gms. of thiourea was added, and the reaction mixture (a colorless solution) stirred overnight at room temperature. There were thus formed the isothiouronium salts of the formulas Without isolating these salts and after cooling in an ice-bath,

Volatile materials were removed in vacuo at 40 C., and finally at C./ 1 mm. The yellow residue was dissolved in a mixture of chloroform and water, the aqueous layer extracted with chloroform, and the combined chloroform extracts were washed twice with water and dried over anhydrous sodium sulfate. Removal of the solvent in vacuo gave a colorless amorphous residue (6.48 gms.). Thin-layer chromatography (1 acetonezl Skellysolve B) showed a major zone of product with a small zone of slightly higher 11,.

This material was chromatographed on silica gel (1.2 kilos, column dimensions 5.8 X cms.) in the system 1 acetone:1.5 Skellysolve B. After a 500 cc. forerun, 50 fractions were collected automatically, and elution of materials followed by thin-layer chromatography. Fractions numbers -173, inclusive, corresponded to the material of higher R,, numbers 185-310, inclusive, corresponded to the major product, and numbers 174-184, inclusive, were a mixture of the two.

Removal of solvent in vacuo from combined fractions 145-173, inclusive, gave a colorless syrup (570 mgms.), which on crystallization from ethyl acetate-Skellysolve B yielded methyl N-acetyl-2,3 ,4-tri-O-'acetyl-7-O-methyl- 1 -thio-a-lincosaminide in small colorless prisms, m.p. 212-213 C. undepressed on a mixture with the sample of Example 31( Part 31 C), of m.p. 21 l213, and also indistinguishable from it by infrared, nuclear magnetic resonance, and mass spectra, and also by optical rotation.

Removal of solvent in vacuo from combined fractions 185-310, inclusive, gave a slightly yellow amorphous solid (4.23 gms.), which on crystallization yielded methyl N-acetyl- 2,3,4-tri-O-acetyl-7-O-methyl-1-thio-fl-lincosaminide in colorless prisms, m.p. l87-188 C.

Anal. Calcd. for C H O NS:

C, 49.64; H, 6.71; N, 3.22; S, 7.36; MeO, 7.13.

Found:

C, 49.73; H, 6.95; N, 3.18; S, 7.64; MeO, 7.41.

[01],, +24 (0, 0.7484, CHCl (mass spec., M') 435 The overall yield of introduction of the SMe group (i.e. a B-anomers) was 49.2 percent (6.7 percent a, 42.5 percent B) with the a/B ratio 1:6.35.

The .B-anomer can be recycled to Part 30-13 and thus enhance the overall yield of the more desired a-anomer.

Part 30-C:

The procedure of Part 30-B was repeated substituting the methylformamide by hexamethylphosphoric triamide (Me,N) P 0) giving an overall yield of 65.5 percent (22.7 percent a, 42.8 percent B) and thus an a/B ratio of 1:1.9.

Part 30-D-1:

Methyl 70-methyl-1-thio-a-lincosaminide (XLVI) NIL- The methyl 7-O-methyl-1-thio-a-lincosaminide-tetraacetate (1.46 gms.) was dissolved in 50 ccs. of hydrazine hydrate and heated under gentle reflux in an oil-bath at C. for 24 hours. Volatile solvent was then removed as completely as possible by distillation at 110 C./15 mm., giving a colorless crystalline residue which was triturated with anhydrous acetonitrile. The solid was removed by filtration and dried. 0n crystallization from a concentrate of 95 percent ethanol, 430 mgs. of methyl 7-0-methyll -thio-a-lincosaminide hemihydrate (Polymorph 1) were obtained as colorless flattened needles, m.p. l26-126.5 C.

Anal. Calcd. for C, H,,0,HS-1/2l-l,0:

C, 43.46; H, 8.03, N, 5.07; S, 11.60; OMe, 11.23; M.W. (anhydrous) 267.35.

XLVI

crystallization was effected slowly in a more dilute solution in 10 95 percent ethanol. Methyl 7-O-methyl--thio-a-lincosaminide hemihydrate was obtained as colorless platelets, m.p. 162l6 3 C. (Polymorph 11).

Both polymorphic forms showed identical chromatographic behavior (R, 0.2 on silica gel TLC in l methanol: 15 chloroform by volume). A mixture melting point of forms 1 and 11 gave the following:

land ll m.p. l26l26.5C. II and ll m.p. l62l63 C. land 11 m.p. l62163 C.

Thus in the presence of Form 11, Form I is converted to Form 11 at some temperature below 162 C. Part 30-E: 7-O-Methyllincomycin hydrochloride (XLVll) CH3 IL- CHa @Miifi l ll HO H01 J)/ SCHa H XLVII A mixture of 3.08 gms. of 4-trans-propylhygric acid hydrochloride and 75 ccs. of acetonitrile was stirred magnetically in a three-necked, 500 cc. flask, equipped with a drying tube and a thermometer extending below the liquid surface. On addition of 3.31 gms. of triethylamine, the solid dissolved rapidly to give a pale tan solution.

On cooling to -5 C. in an ice/methanol bath, a colorless precipitate of triethylammonium chloride separated. Without removal of the precipitate, 2.02 gms. (1.94 ccs.) of isobutyl chloroformate were added at such a rate that the temperature remained between 5 and 8 C., after which stirring was continued at -5 C. for 15 minutes.

There were then rapidly added 2.0 gms. of methyl 7-0- methyl-1-thio-a-lincosaminide in 25 ccs. of water to the above mixed anhydride solution, giving a pale tan solution, which was stirred at C. for 45 minutes. Thin-layer chromatography (silica gel, 8 ethyl acetatez acetonezl water by volume) showed a trace only of residual aminosugar, and a major new zone of R,= 0.4. Volatile solvent was removed in vacuo at 40 C., the tan aqueous residual solution adjusted to pH 10 by the addition of aqueous sodium hydroxide (N), the mixture extracted thrice with 100 cc. portions of chloroform, and the combined extracts washed with water and dried over anhydrous sodium sulfate. Removal of the solvent in vacuo at 40 C. gives a tan amorphous solid (2.32 gms.).

Chromatography on silica gel (450 gms., column dimensions 3.8 X 95 cms.) in the system 1 methanol: chloroform by volume following a forerun (250 ccs.) after which cc. fractions were collected automatically, gave 7-O-methyllincomycin in fractions 44-70, inclusive, obtained on removal of the solvent in vacuo as a colorless syrup (2.20 gms.). This syrup was dissolved in water (5 ccs.) by stirring and adding hydrochloric acid (concentrated) to attain a pH of 3, the solution filtered under suction, the sinter washed with water (3 ccs.) and the filtrate and washings cooled in an ice-methanol bath. With stirring, acetone (200 ccs.) was added, followed by ether 100 ccs. giving a colorless crystalline precipitate which was collected and dried in a vacuum desiccator at room temperature. The solid (1.71 gms.) was obtained as small, elongated, colorless platelets, m.p. l55l 57 C.

Anal. Calcd. for C,,,H O N S-HCl: c, 49.93; H, 8.16; N, 6.13; S, 7.02; Cl, 7.76; OMe, 6.79

M.W. (free base) 420.57.

Found (corrected for 4.83 percent H O);

C, 50.09; H, 8.22; N, 6.02; S, 7.20; Cl, 7.46; OMe, 7.03

[11],, +145C (c, 1.063, H O) pKa' 7.6

M01. Wt.:

(mass spec., M of free base) 420 The 7(R)-O-methyllincomycin thus produced can be further processed by the novel process of this invention to yield the corresponding 3-nucleotide.

EXAMPLE 31 Part 31-A-l:

Methyl N-acetyl-2-O-acetyl-3,4-O isopropylidenel-thio-alincosaminide (XLVIII) ll CHaC-NH- 0A0 XLVIII Methyl 6-N,7-O-ethylidyne3 ,4-0-isopropylidene- 1 -thio -alincosaminide (5gms.) (Example l C of U.S. Pat. No. 3,337,527) was acetylated by leaving overnight at room temperature in a mixture of pyridine (25 ccs.) and acetic anhydride 12 ccs). Removal of solvent on a rotating evaporator in vacuo at 40 C. gave a pale yellow syrup which was dissolved in chloroform, washed with water, saturated aqueous sodium bicarbonate, againwith water, and dried over anhydrous sodium sulfate. Thin-layer chromatography (silica gel, 75 methylethyl ketone;25 acetonezlO water by volume) showed the absence of starting material, and the formation of a new zone of slightly higher R Removal of the solvent in vacuo at 40 C. gave methyl 2-O-acetyl-6N, 7-O-ethylidyne- 3,4-O-isopropylidene-l-thio-a-lincosaminide as an almost colorless syrup which could not be induced to crystallize.

Water (75 ccs.) at pH 7 was added and, with magnetic stirring, the mixture was heated on asteam-bath. After 6 hours the solvent was removed in vacuo at 40 C. to give a colorless crystalline solid (5.95 gms.) which was chromatographed on silica (600 gms., column dimensions 4.8 X 79 cms.) in the system 1 methanol:7 chloroform (by volume). After a 650 cc. forerun, 25 cc. fractions were collected automatically, the elution being followed by thin-layer chromatography. The desired material was present in fractions 35-41, inclusive. Removal of the solvent gave a colorless amorphous solid 1 .57 gms.). Recrystallization from acetone-Skellysolve B (technical hexane) gave colorless needles of methyl N-acetyl-2-O- acetyl-3,4-O-isopropylidine-l-thioa-lincosaminide, m.p. l78179 C.

Anal. Calcd. for C, H O NS:

C, 50.92; H, 7.21; N, 3.71; S, 8.49; N.W. 377.46.

Found:

C, 50.50; H, 7.20; N, 3.77; S, 8.50.

[a],,+194(c, 0.7342, CHCl Mol. Wt.:

(mass spec. M*) 377 Part 31-A-2:

The procedure of Part 3l-A-1 was repeated except that the solvent was removed after a heating time of 2 hours (instead of 6 hours). The yield of methyl N-acetyl-2-Oacetyl- 3,4-O-isopropylidene-l-thio-a-lincosaminide was increased to 60.5 percent.

Part 3 l-B:

Methyl N-acetyl-Z-O-acetyl-7-O-methyl-3,4-O-isopropylidenel-thio-a-lincosaminide (XLVlX) O CHaA-NH A0 xnvrv Methyl N-acetyl-2-0-acetyl-3,4-O-isopropylidene-l-thio-alincosaminide (1.0 gm., 1 mol.), methyl iodide (37.6 gms., 16.5 ccs., 100 mols.), and silver oxide (3.1 gms., 5 mols.) were heated and stirred under gentle reflux for 16 hours. The [5 methyl iodide was removed in vacuo at 40 C., and the resulting yellow-gray powder was extracted thoroughly with methylene chloride. Removal of the solvent in vacuo gave a yellow syrup (1.09 gms.). This crude product was subjected to countercurrent distribution (500 transfers) in the system 1 ethyl acetatezl ethanolzl water:-2 cyclohexane, by volume, using equal volumes of upper and lower phaserA major peak was found, of K 0.34, matching the theoretical curve.

Removal of the solvent from the combined fractions of the material of K 0.34 yielded a syrup (250 mgms.) which crystallized on standing. Recrystallization from ethyl acetate- Skellysolve B, gave methyl N-acetyl-2-O-acetyl-7-O-methyl- 3,4-O-isopropylidene-l-thio-a-lincosaminide as blunt, colorless needles m.p. 152154 C. (160 mgms.). A second recrystallization from the same solvent mixture yielded the 30 pure product, m.p. l52.5-l 54 C.

Anal. Calcd. for C,-,H ,O-,NS: C, 52.15; H, 7.47; N, 3.58; S, 8.19; N.W. 391.48. Found: C, 52.24; H, 7.48; N, 3.92; S, 7.98. 3 Mol. Wt.: (mass spec, M") 391 [a],, +188(c, 1.185, CHCI,) Part 31-C: 7

Methyl N-acetyl-7-O-methyl-l-thio-a-lincosaminide and its 40 triacetate Methyl N-acetyl-2-O-acetyl-3,4-0-isopropylidene-l-thio-alincosaminide (100 mgms.) was stirred with water (20. ccs.) and aqueous hydrochloric acid (N, 5 ccs.) at roomtemperature overnight. The solution was neutralized by stirring with silver carbonate (3 gms.), the solids removed by filtration and washed with water, and the filtrate and washings taken to dryness on a rotating evaporator at 6017 mm., giving methylN- acetyl-7-O-methyl-1-thio-a-lincosaminide as a colorless syrup which did not crystallize. It was further characterized by converting it to the triacetate. Y

Pyridine (5 ccs.) and acetic anhydride (3 ccs.) wereadded, the mixture swirled till the syrup had dissolved, and the mixture left overnight at room temperature. Solvent was then removed as completely as possible at 40 C.l l mm., giving av tan crystalline mixture, which was dissolved in chlorofonn, washed with aqueous hydrochloric acid (N/l0), water, saturated 'aqueous sodium bicarbonate, water, and dried over anhydrous sodium sulfate. Removal of the solvent in vacuo gave methyl N-acetyl-2,3,4-tri-O-acetyl-7-O-methyl -l-thio-a-lincosaminide as an almost colorless crystalline solid which separated from ethyl acetate-Skellysolve B in small colorless prisms, m.p. 21 1.5213C.

Anal. Calcd. for C,,H,,O,NS:

C, 49.64; H, 6.71; N, 3.22; S, 7.36; MeO, 7.13; N.W. v

wherein R, is hydrogen or lower alkyl, for example, methyl and ethyl, and R is hydrogen or lower alkyl, for example,

- methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl,

and the isomeric forms thereof, or lower cycloalkyl, for example, cyclopentyl, cyclohexyl, and cyclohexylmethyl to give compounds of the following formula wherein R, and R are as given above. The resulting lincomycin compounds can be converted to the corresponding novel 3-nucleotides by the novel processes of this invention.

We claim: 1. A compound of the formula:

and the pharmacologically acceptable salts thereof, wherein Y is -SR wherein R is alkyl of one to six carbon atoms, inclusive;

; -S--CH,' CH,OH; or OR wherein R is alkyl of from one to eight carbon atoms, inclusive, and isomeric forms thereof;

R is H or cis or trans lower-alkyl of from one to eight carbon atoms; R, is H, CH, or C,l-l,; X is OH, chlorine, bromine, iodine or OR,, wherein R is alltyl of one to six carbon atoms, inclusive, each in the (R) or (S) configuration; and Z is a nucleoside-5'-phosphate group wherein said nucleoside is adenosine, guanosine, cytidine or uridine.

2. The zwitterion form of the compound of claim 1.

3. A compound according to claim 1 having the fonnula: 

2. The zwitterion form of the compound of claim
 1. 2. absorbing the resulting filtrate on a suitable absorbent to remove water-soluble impurities;
 3. chromatographing the resulting eluate from the absorbent on an anion exchange resin;
 3. A compound according to claim 1 having the formula:
 4. A compound according to claim 3 having the formuLa:
 4. subjecting fractions from the anion exchange resin to counter current distribution; and
 5. separating the individual 3-nucleotides by chromatography.
 5. A compound according to claim 3 having the formula:
 6. A compound according to claim 1 having the formula:
 7. A compound according to claim 6 having the formula:
 8. A compound according to claim 6 having the formula:
 9. A compound according to claim 1 having the formula:
 10. A compound according to claim 9 having the formula:
 11. A compound according to claim 9 having the formula:
 12. A compound according to claim 1 having the formula:
 13. A compound according to claim 12 having the formula:
 14. A compound according to claim 12 having the formula:
 15. A compound according to claim 1 wherein Y is -SCH3, Rl is propyl, R2 is CH3, X is chlorine, and Z is a nucleoside-5''-phosphate group wherein said nucleoside is cytidine.
 16. A compound according to claim 1 wherein Y is -SCH3, R1 is propyl, R2 is CH3, X is chlorine, and Z is a nucleoside-5''-phosphate group wherein said nucleoside is adenosine.
 17. A compound according to claim 1 wherein Y is -SCH3, R1 is propyl, R2 is CH3, X is chlorine, and Z is a nucleoside-5''-phosphate group wherein said nucleoside is uridine.
 18. A compound according to claim 1 wherein Y is -SCH3, R1 is propyl, R2 is CH3, X is chlorine, and Z is a nucleoside-5''-phosphate group wherein said nucleoside is guanosine.
 19. A process for isolating a compound of the formula: 